含微纳结构且二级结构可控的丝蛋白支架的研究
发布时间：2019-07-01 13:24
【摘要】：过去十年，由于其优良的生物相容性、生物可降解性、低炎症反应和优秀的机械性能，丝蛋白支架材料已经从传统的纺织纤维材料转变成具有普适意义的天然生物材料和组织修复材料。但是不同组织的修复对材料有着不同的特定要求，如何进一步提高支架的生物相容性和诱导性使其更有利于不同组织的修复是我们面临的一项挑战。细胞外基质（ECM）的微纳结构为支架的设计包括支架多孔结构的设计提供了良好的模板，通过模拟ECM的微纳结构构建有利于细胞和组织生长的微环境为构建具有生物活性的组织修复载体提供了全新的方向。 本文首先阐明了丝蛋白在水溶液环境下可控自组装形成不同微纳结构对支架材料成孔性的调控机制，随后研究了粘度、二级结构和水作用力对成孔性的调控作用。实验发现，溶液中纳米线的形成对支架在冻干过程中多孔结构的形成具有关键作用，是支架材料成孔的关键因素。 在此基础上，本研究通过缓慢浓缩处理的方法自组装形成纳米纤维，并通过冷冻干燥法获得具有仿细胞外基质纳米纤维结构的丝蛋白多孔支架。经上述方法制备的支架孔径在200~250um之间、孔隙率达到99%以上，且随着丝蛋白的逐渐降解和溶解，纳米纤维会暴露到外部，在细胞体内外培养中有利于细胞的吸附、增殖和迁移。随后利用甲醇和水处理等不同方法实现对多孔支架晶体结构的调控，使得支架材料具有不同的稳定性和降解行为，以满足不同组织再生的具体要求。其中，当纯水蒸气处理时丝素的结晶结构由无规向Silk I结构转变，随着甲醇含量增多，丝素的结晶结构由Silk I结构逐渐转变为Silk II结构，热力学稳定性增强，降解速率降低，扩大了其在组织工程的不同应用。 最后，通过体外细胞培养，用盐析法制备的丝蛋白多孔支架作为对比，研究了该多孔材料对大鼠骨髓间充质干细胞粘附、增殖的影响。激光共聚焦显微镜和DNA含量结果表明，同盐析法丝蛋白支架相比，本研究所制备的仿生纳米纤维化丝蛋白多孔支架对BMSC细胞的生长和增殖具有显著的促进作用。 综上所述，，本文通过丝蛋白自组装技术制备出孔结构和二级结构可控的含有仿生纳米纤维结构的丝蛋白多孔材料，为组织工程或组织修复提供一种具有生物活性的支架材料，可望满足不同组织再生的要求和应用。
[Abstract]:In the past decade, due to its excellent biocompatibility, biodegradability, low inflammatory reaction and excellent mechanical properties, silk protein scaffold materials have changed from traditional textile fiber materials to natural biomaterials and tissue repair materials with universal significance. However, the repair of different tissues has different specific requirements for materials. How to further improve the biocompatibility and inductivity of scaffolds to make them more conducive to the repair of different tissues is a challenge we face. The micro-nano structure of extracellular matrix (ECM) provides a good template for the design of scaffolds, including the design of porous structure of scaffolds. By simulating the micro-nano structure of ECM, the construction of microenvironment conducive to cell and tissue growth provides a new direction for the construction of bioactive tissue repair vector. In this paper, the regulation mechanism of different microstructures formed by controllable self-assembly of silk proteins in aqueous solution on the porosity of scaffolds was studied, and then the effects of viscosity, secondary structure and water force on the porosity of scaffolds were studied. It is found that the formation of nanowires in solution plays a key role in the formation of porous structure of scaffolds during freeze-drying, and is the key factor for pore formation of scaffolds. On this basis, the nanofibers were self-assembled by slow concentration treatment, and the silk protein porous scaffolds with extracellular matrix nanofiber structure were obtained by freeze-drying method. The pore size of the scaffold prepared by the above method is between 200~250um and the porosity is more than 99%. With the gradual degradation and dissolution of silk protein, the nanofibers will be exposed to the outside, which is beneficial to the adsorption, proliferation and migration of cells in vitro and in vivo. Then different methods such as methanol and water treatment are used to regulate the crystal structure of porous scaffolds, which makes the scaffolds have different stability and degradation behavior to meet the specific requirements of different tissue regeneration. Among them, when pure water vapor treatment, the crystal structure of silk fibroin changes from random to Silk I structure. With the increase of methanol content, the crystal structure of silk fibroin gradually changes from Silk I structure to Silk II structure, thermodynamic stability increases, degradation rate decreases, and its different applications in tissue engineering are expanded. Finally, the effect of porous silk protein scaffold prepared by salting out method on the adhesion and proliferation of rat bone marrow mesenchymal stem cells (BMSCs) was studied by cell culture in vitro. The results of laser confocal microscope and DNA content showed that the biomimetic nano-fibrosis silk protein porous scaffolds prepared in this study could significantly promote the growth and proliferation of BMSC cells compared with salting out silk protein scaffolds. In summary, silk protein porous materials with controllable pore structure and secondary structure containing biomimetic nanofiber structure were prepared by silk protein self-assembly technique, which provides a bioactive scaffold material for tissue engineering or tissue repair, which is expected to meet the requirements and applications of different tissue regeneration.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08
本文编号：2508515
[Abstract]:In the past decade, due to its excellent biocompatibility, biodegradability, low inflammatory reaction and excellent mechanical properties, silk protein scaffold materials have changed from traditional textile fiber materials to natural biomaterials and tissue repair materials with universal significance. However, the repair of different tissues has different specific requirements for materials. How to further improve the biocompatibility and inductivity of scaffolds to make them more conducive to the repair of different tissues is a challenge we face. The micro-nano structure of extracellular matrix (ECM) provides a good template for the design of scaffolds, including the design of porous structure of scaffolds. By simulating the micro-nano structure of ECM, the construction of microenvironment conducive to cell and tissue growth provides a new direction for the construction of bioactive tissue repair vector. In this paper, the regulation mechanism of different microstructures formed by controllable self-assembly of silk proteins in aqueous solution on the porosity of scaffolds was studied, and then the effects of viscosity, secondary structure and water force on the porosity of scaffolds were studied. It is found that the formation of nanowires in solution plays a key role in the formation of porous structure of scaffolds during freeze-drying, and is the key factor for pore formation of scaffolds. On this basis, the nanofibers were self-assembled by slow concentration treatment, and the silk protein porous scaffolds with extracellular matrix nanofiber structure were obtained by freeze-drying method. The pore size of the scaffold prepared by the above method is between 200~250um and the porosity is more than 99%. With the gradual degradation and dissolution of silk protein, the nanofibers will be exposed to the outside, which is beneficial to the adsorption, proliferation and migration of cells in vitro and in vivo. Then different methods such as methanol and water treatment are used to regulate the crystal structure of porous scaffolds, which makes the scaffolds have different stability and degradation behavior to meet the specific requirements of different tissue regeneration. Among them, when pure water vapor treatment, the crystal structure of silk fibroin changes from random to Silk I structure. With the increase of methanol content, the crystal structure of silk fibroin gradually changes from Silk I structure to Silk II structure, thermodynamic stability increases, degradation rate decreases, and its different applications in tissue engineering are expanded. Finally, the effect of porous silk protein scaffold prepared by salting out method on the adhesion and proliferation of rat bone marrow mesenchymal stem cells (BMSCs) was studied by cell culture in vitro. The results of laser confocal microscope and DNA content showed that the biomimetic nano-fibrosis silk protein porous scaffolds prepared in this study could significantly promote the growth and proliferation of BMSC cells compared with salting out silk protein scaffolds. In summary, silk protein porous materials with controllable pore structure and secondary structure containing biomimetic nanofiber structure were prepared by silk protein self-assembly technique, which provides a bioactive scaffold material for tissue engineering or tissue repair, which is expected to meet the requirements and applications of different tissue regeneration.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08
【参考文献】
相关期刊论文 前4条
1 王海宁;万怡灶;李建;张胜男;何芳;黄远;王玉林;;纳米纤维组织工程支架及其纳米效应研究进展[J];材料导报;2007年04期
2 徐军;纳米材料的研究现状与发展[J];电镀与涂饰;2001年06期
3 李明忠,吴徵宇,卢神州,张长胜,严灏景;多孔丝素材料的结构与性能研究[J];东华大学学报(自然科学版);2001年02期
4 王身国;组织工程细胞支架及其相关技术研究[J];现代康复;2001年16期
本文编号：2508515
本文链接：https://www.wllwen.com/yixuelunwen/swyx/2508515.html
